OSBS Kinetics Asssay for SpectraMax Plate Reader
Last Edited by Andy McMillan 1-15-2015
A. Measure Substrate Concentration and Pathlength
a. Make set of serial dilution appropriate for Km of enzyme.
i. Max measureable concentration on SpectraMaxPlus 384 using plate is
~1500 uM, Max for the cuvette is ~1000 uM. Because of difference make
stock at 5x concentration for assay, and use 10x for measuring substrate
concentration
ii. Minimum is ~10 uM using the plate reader
b. Measure absorbance of each substrate concentration in the cuvette using a 1:10
dilution
c. Measure absorbance of each concentration on plate reader. Make sure path
length correction is not being used. Volume should be the same as using for
assay (100 uL)
d. Pathlength is Aplate/Acuvette. Value should be ~0.3
e. Calculate substrate concentration of substrate using Beer’s Law (Concentration =
Absorbance/(*pathlength=2400 M-1cm-1 at 310 nm and 4000 M-1cm-1 at 293
nm. Make sure correcting for uM vs M units in extinction coefficient
B. Measure Protein Concentration
a. Spin down some of protein stock in centrifuge remove any aggregates
b. Measure concentration in cuvette by absorbance at 280 nm, as well as A330 to
correct for light scattering.
Acorrected = A280- (1.928*A330)
c. Measure at several different dilutions, for typical protein expression levels I find
1:20, 1:10 and 1:5 work.
d. If needed dilute with storage buffer+glycerol and repeat concentration
measurement (see below for how mixing reaction).
C. Kinetics Runs
a. Make 5 mL buffer mix of Tris, MnCl2 and water.
i. 0.1 mM MnCl2 (625 uL from 1 mM stock)
ii. 50 mM Tris pH 8.0 (625 uL from 500 mM stock)
iii. Water to 3750 uL minus enzyme that will be needed
iv. Split 1 mL out and add enzyme volume equivalent of water, this will be
used for blank reaction
b. For each reaction:
i. Make sure plate setup is correct in software.
1. Usually read every 3s for 30 s. Adjust settings if needed for
different proteins.
2. Read at 310 nm (293 would give more signal, but could be
influenced by absorbance by Trp and Tyr in enzyme
3. Change plate read area and plate layout for wells used in this run.
a. Samples should all be in the same column, so can be read
at the same time
b. Can be changed quicker by copying plate, change read
area to include both well strips, copy layout for one strip
into new one, then change read area back to single strip
ii. Add 1 mL of buffer mix to center well of plastic tray, add enzyme (amount
needed for good signal varies, but wildtype tends to be 0.02), transfer
400 uL to second well.
iii. Add blank mix to third well, can be transferred between trays for each
replicate.
iv. Add 20 uL of substrate to each well, as well as water for blank.
v. Start reaction with p300 multichannel pipet by transferring 80 uL of mix
to appropriate well. Mix ~6 times, but take care not to create bubbles.
vi. Start recording with software
vii. SAVE DATA
D. Data Analysis (Can be done during/between runs)
a. Copy data from run into spreadsheet. (Fits can be done in plate reader software,
but only able to fit whole dataset over the same interval)
b. Fit data to from beginning of reaction to where rate begins to change
i. Frequently there’s some lag at the beginning. If it’s just the first point or
two, this may be ok to leave out of fitting, otherwise mixing more
vigorously could be needed.
ii. Changing format of time column and explicitly putting in seconds value is
needed for proper value of fit.
iii. Make sure fit uses enough decimal places, this will probably require
changing to scientific notation
c. Convert rate from AU/s to uM/s using
C = Al
=2400
and l = pathlength
d. Get kobs by dividing by enzyme concentration
e. Plot kobs vs substrate concentration in Kaleidograph
i. Fit to Michelis-Menton equation (should be already defined) to
determine kcat and Km
M-1cm-1
ii. If not reaching saturation fit low concentrations to a linear plot for
kcat/Km value
E. Cleanup
a. Wash cuvette as soon as done measuring concentrations, place in case and keep
in drawer by plate reader
b. Wash plates immediately after done. Plates can be reused if cleaned properly,
but if dry out tend to leave discoloration that is difficult to remove.
c. Empty tip container, this assay goes through a lot of tips, and beaker is usually
almost full afterwards.
d. MAKE SURE PLATE READER DRAWER IS CLOSED AND MACHINE IS TURNED OFF
BEFORE LEAVING